Hepatitis C virus is a chronic, viral disease that infects the liver. Infection is transmitted by injection drug use or contaminated blood products, and approximately 80% of infected individuals become chronically infected. Chronic HCV infection is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. Indeed, HCV is the leading cause of liver transplantation in the United States. Currently, there is no vaccine for HCV and the treatment regimen, pegylated Interferon-alpha2b and ribavirin, is only effective in ~50% of patients. Due to a lack of small animal model and, until recently, a cell culture adapted virus, little is known about the immune response against HCV infection. HCV Envelope-2 (E2) is expressed on the surface of the virion and is thought to be important in mediating viral attachment and entry. Neutralizing antibodies against the E2 protein are found in infected patients, although the mechanisms by which these antibodies act and the epitopes to which they bind remains poorly understood. An improved understanding of how anti-E2 antibodies neutralize infection and identification of the important neutralizing epitopes would provide a framework for the future development of vaccines and novel therapeutics against HCV. The objective of this proposal is to study the mechanism of antibody neutralization directed against HCV E2 and to identify the antibody binding regions within the protein that are important in controlling infection. To achieve this objective, we propose two specific aims. In the first aim we propose to test the neutralization capabilities of a large panel of anti-E2 antibodies in vitro. We will test the ability of HCV E2 antibodies to interfere with different phases of the viral life cycle, to block infection of viral particles of different densities, and to interact with the complement system to enhance neutralization of infection. This aim will enable us to better understand how antibodies function to limit HCV infection. In our second aim, we propose to investigate the molecular mechanism of antibody-mediated neutralization of HCV E2. In this aim we plan to map the critical antibody binding residues within E2 and to test antibodies for their ability to inhibit HCV binding to CD81. This will provide a more thorough understanding of the critical residues within E2 that are necessary for viral entry. Together, these aims will allow us to generate a better understanding of how HCV neutralizing antibodies inhibit infection and whether specific viral epitopes can be used as targets for vaccine development or the generation of novel therapeutics.